Human cd3-specific antibody with immunosuppressive properties

ABSTRACT

Described are mono- and multivalent scFv-antibodies comprising the binding sites specific for human T cell marker CD 3 . These antibodies are strongly immunosuppressive and do not cause a significant release of cytokines. Furthermore, polynucleotides encoding said antibodies are described as well as vectors comprising said polynucleotides, host cells transformed therewith and their use in the production of said antibodies. Pharmaceutical compositions containing any of the above mentioned polynucleotides, antibodies or vectors are useful for immunotherapy, preferably against acute transplant rejections.

The present invention relates to mono- and multivalent scFv-antibodiescomprising the binding sites specific for the human T cell marker CD3.The antibodies of the invention are strongly immunosuppressive and donot cause a significant release of cytokines. The present invention alsorelates to polynucleotides encoding said antibodies as well as vectorscomprising said polynucleotides, host cells transformed therewith andtheir use in the production of said antibodies. Finally, the presentinvention relates to compositions, preferably pharmaceuticalcompositions, comprising any of the above mentioned polynucleotides,antibodies or vectors. The pharmaceutical compositions are useful forimmunotherapy, preferably against acute transplant rejections.

OKT3, a murine IgG2a mAb directed against the ε-chain of the CD3 complexon human T lymphocytes (Salmeron et al., J. Immunol. 147 (1991),3047-3052) and produced by a hybridoma with the ATCC deposit number ofCRL 8001 is used to prevent tissue rejection after renal and hepatictransplantation, and provides an alternative treatment for transplantrejections that are unresponsive to corticosteroids. In vivo,administration of OKT3 induces a dramatic decrease in the number ofcirculating CD3⁺cells as it down-modulates the T-cell receptor (TCR).However, adverse effects can occur during the first days of treatment.Chills and fever often follow the administration of OKT3 and patientsoccasionally suffer from nausea, vomiting, diarrhea, dyspnea, wheezing,and sterile meningitis. Many of these side effects have been attributedto the release of cytokines, especially from T cells. After a moreprolonged period of use, many patients develop a human anti-mouseantibody (HAMA) response.

Binding of OKT3 alone is insufficient to trigger T cells. Proliferationof T cells which induces the release of cytokines like IL-2, IL-6,TNF-αand IFN-γ results from cross-linking of T cells and FcR-bearingcells. Human IgG Fc receptors (FcγRI, FcγRII, FcγRIII) are distributedon human monocytes/macrophages, B lymphocytes, NK cells andgranulocytes. They all bind to the C_(H)2 region of both mouse and humanIgG, differing in their affinity. The immunogenicity of such anti-CD3 Abhas been reduced by using chimeric antibodies made from the variabledomains of a mouse mAb and the constant regions of a human Ab. To reducebinding to Fc receptors, Fc domains from particular classes of human IgGhave been employed or mutations have been introduced into the Fc domainin the parts that bind to the Fc receptors. However, interactions of theFc domains cannot be completely abrogated and the efficacy of theimmunosuppressive activity was not increased.

Thus, the technical problem underlying the present invention was toprovide means more suitable for preventing allograft rejection thatovercome the disadvantages of the means of the prior art.

The solution of the said technical problem is achieved by providing theembodiments characterized in the claims. Antibodies have beenconstructed that are more efficient in suppressing T cell activation andproliferation by down-regulating the CD3 molecule but that do not causea large release of cytokines, thus avoiding many of the unpleasantside-effects. These antibodies only comprise the variable immunoglobulindomains, so called F_(v) modules by means of which undesired immuneresponses can be avoided. The F_(v) module is formed by association ofthe immunoglobulin heavy and light chain variable domains, V_(H) andV_(L), respectively. Preferred embodiments of these antibodies are basedonly on the variable domains of the OKT3 antibody, but contain a serineinstead of a cysteine at position H100A of the heavy chain (according tothe Kabat numbering system). This mutation has previously been shown toimprove the stability of the single chain Fv molecule (Kipriyanov etal., Protein Engineering 10 (1997), 445-453). Surprisingly, suchantibodies, and in particular a bivalent antibody in a so-called diabodyformat, had a much greater immunosuppressive effect as measured by CD3downregulation and inhibition of T cell proliferation in a mixedlymphocyte reaction (MLR) than the original parental OKT3 antibody and,in contrast to the parental OKT3, caused no significant release of thecytokines IFN-α and IL-2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of mono- and multivalent single chainFv-antibody constructs

Diabody: non-covalent scFv dimer; scDb: single chain diabody; scFv:single chain Fv fragment; (scFv)₂: scFv-scFv dimer. The antibody V_(H)and V_(L) domains are shown as black and gray ovals, respectively.

FIG. 2: Expression cassettes for anti-CD3 scFv constructs

His₆: six C-terminal histidine residues; L: short peptide linker (theamino acid sequence is shown in bold) connecting the V_(H) and V_(L)domains; leader, bacterial leader sequence (e.g. PelB leader) forsecretion of recombinant product into periplasm; rbs, ribosome bindingsite; Stop: stop codon (TAA); V_(H) and V_(L): variable regions of theheavy and light chains specific to human CD3. Four C-terminal aminoacids of V_(H) domain and four N-terminal amino acids of the V_(L)domain are underlined.

FIG. 3: Diagram of the expression plasmid pSKK3-scFv6_(—OKT)3

bla: gene of beta-lactamase responsible for ampicillin resistance; bp:base pairs; CDR-H1, CDR-H2 and CDR-H3: sequence encoding thecomplementarity determining regions (CDR) 1-3 of the heavy chain;CDR-L1, CDR-L1, CDR-L2 and CDR-L3: sequence encoding the complementaritydetermining regions (CDR) 1-3 of the light chain; CH1-L6 linker:sequence which encodes the 6 amino acid peptide Ser-Ala-Lys-Thr-Thr-Proconnecting the V_(H) and V_(L) domains; His6 tag: sequence encoding sixC-terminal histidine residues; hok-sok: plasmid stabilizing DNA locus;lacI: gene encoding lac-repressor; lac P/O: wild-type lac-operonpromoter/operator; M13ori: intergenic region of bacteriophage M13;pBR322ori: origin of the DNA replication; PelB leader: signal peptidesequence of the bacterial pectate lyase; rbs1: ribosome binding sitederived from E. coli lacZ gene (lacZ); rbs2 and rbs3: ribosome bindingsite derived from the strongly expressed gene 10 of bacteriophage T7(T7g10); skp gene: gene encoding bacterial periplasmic factor Skp/OmpH;tHP: strong transcriptional terminator; tLPP: lipoprotein terminator oftranscription; V_(H) and V_(L): sequence coding for the variable regionof the immunoglobulin heavy and light chain, respectively. Uniquerestriction sites are indicated.

FIG. 4: Analysis of purified anti-CD3 scFv antibodies by 12% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) underreducing conditions

Lane 1: Mr markers (kDa, M_(r) in thousands); Lane 2: anti-CD3 scFv₁₀;Lane 3: anti-CD3 scFv₆. The gel was stained with Coomassie Blue.

FIG. 5: Analysis of purified anti-CD3 scFv antibodies by size exclusionchromatography on a calibrated Superdex 200 column

The elution positions of molecular mass standards are indicated.

FIG. 6: Lineweaver-Burk analysis of fluorescence dependence on antibodyconcentration as determined by flow cytometry Binding of mAb OKT3(circles), scFv₆ (triangles) and scFv₁₀ (squares) to CD3⁺Jurkat cellswas measured.

FIG. 7: Retention of anti-CD3 antibodies on the surface of CD3⁺Jurkatcells at 37° C.

Cell-surface retention of mAb OKT3 (circles), scFv₆ (triangles) andscFv₁₀ (squares) on CD3⁺Jurkat cells was measured. Values are expressedas a percentage of initial mean fluorescence intensity.

FIG. 8: Proliferation of peripheral blood mononuclear cells (PBMC) after24 h incubation in presence of mAb OKT3 and anti-CD3 scFv-antibodies atconcentrations of 0.01-10 μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in microtiter plates at densityof 2×10⁵ cells/well either without antibodies or in presence of serialdilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀ . After 24 hincubation, the cells were pulsed with 10 μM BrdU for 18 h.Incorporation of BrdU was determined by BrdU-ELISA. The means and SDs oftriplicates are shown.

FIG. 9: Proliferation of PBMC after 72 h incubation in presence of mAbOKT3 and anti-CD3 scFv-antibodies at concentrations of 0.01-10 μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in microtiter plates at densityof 2×10⁵ cells/well either without antibodies or in presence of serialdilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After 72 hincubation, the cells were pulsed with 10 μM BrdU for 18 h.Incorporation of BrdU was determined by BrdU-ELISA. The means and SDs oftriplicates are shown.

FIG. 10: Release of IL-2 by PBMCs after 24 h incubation in presence ofmAb OKT3 and anti-CD3 scFv-antibodies at concentrations of 0.01-10 μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in 24-well plates at a densityof 2×10⁶ cells/well either without antibodies or in presence of serialdilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After 24 hincubation, samples from the culture supernatants were harvested and theIL-2 concentration was measured by ELISA. The mean values of duplicatesare shown.

FIG. 11: Release of IFN-α by PBMCs after 72 h incubation in presence ofmAb OKT3 and anti-CD3 scFv-antibodies at concentrations of 0.01-10 μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in 24-well plates at a densityof 2×10⁶ cells/well either without antibodies or in presence of serialdilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After 72 hincubation, the samples of culture supernatants were harvested and theconcentration of IFN-α was measured by ELISA. The mean values ofduplicates are shown.

FIG. 12: Release of TNF-α by PBMCs after 36 h incubation in the presenceof mAb OKT3 and anti-CD3 scFv-antibodies at concentrations of 0.01-0.1μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in 24-well plates at a densityof 2×10⁶ cells/well either without antibodies or in presence 0.1 μg/mland 0.01 μg/ml of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After 36h incubation, samples of the culture supernatants were harvested and theconcentration of TNF-α was measured by ELISA. The mean values ofduplicates are shown.

FIG. 13: Induction of the expression of IL-2Rα (CD25) on T cells after90 h incubation of PBMC cultures in presence of mAb OKT3 and anti-CD3scFv-antibodies at concentrations of 0.01-10 μg/ml

PBMCs from healthy donor A or donor B alone and mixed lymphocyte cultureof PBMCs from donor A plus B were seeded in 24-well plates at a densityof 2×10⁶ cells/well either without antibodies or in presence of serialdilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After 90 hincubation, the CD25 expression was detected by flow cytometry usinganti-CD25 mAb B1.49.9. Mean fluorescence intensity values aftersubtracting background fluorescence are shown.

FIG. 14: CD3 modulation and coating by mAb OKT3 and anti-CD3scFv-antibodies

PBMCs from healthy donor A or donor B were seeded in 24-well plates at adensity of 2×10⁶ cells/well either without antibodies or in presence ofserial dilutions of mAb OKT3, anti-CD3 scFv₆ and anti-CD3 scFv₁₀. After24 h incubation, the cells were harvested and stained withFITC-conjugated anti-CD3 mAb OKT3, PC5-conjugated anti-TCRα/β mAbBMA031. T cells were counterstained with anti-CD5 mAb and analyzed byflow cytometry. Data for CD3 modulation represent the percentage ofTCR/CD3 complexes on the surface of treated CD5-positive T cells as afraction of TCR/CD3 complexes on the surface of untreated CD5-positive Tcells. CD3 coating is shown as the fraction of TCR/CD3 complexes whichcould not be detected by FITC-conjugated OKT3.

FIG. 15: (a) DNA sequence of plasmid pSKK3-scFv6 anti-CD3; (b) aminoacid sequence of the V_(H) and V_(L) connected by the peptide linkerSAKTTP encoded by the DNA sequence contained in pSKK3-scFv6 anti-CD3

Thus, the present invention relates to an antibody characterized by thefollowing features:

-   -   (a) it is capable of suppressing an immune reaction;    -   (b) it is devoid of constant antibody regions; and    -   (c) it binds an epitope on the CD3 complex of the T-cell        receptor.

The antibody of the present invention is specific to human TCR/CD3complex present on all T cells regardless their MHC specificity. Suchantibody is capable to suppress the activated T lymphocytes without anysignificant release of inflammatory cytokines, thus avoiding many of theunpleasant side-effects. The release of cytokines, e.g., IL-2, IFN-γ andTNF-α is reduced by a factor more than 100 compared to OKT3. This is insharp contrast with any known immunosuppressive antibodies. Althoughimmunosuppression can be achieved by the administering such traditionalantibodies to humans, their efficacy is often compromised by twofactors: the first-dose syndrome resulting from T-cell activation, andthe anti-globulin response (e.g. HAMA response) resulting from multipleinjections of foreign proteins of non-human origin. The symptoms ofantibody toxicity include fever, chills, diarrhea, and vomiting and insevere cases have resulted in death. The syndrome is caused by therelease of inflammatory cytokines as result of transient T cellactivation. Such activation depends on the interaction of the Fc portionof the antibody and Fc receptors (FcR) on accessory cells to cross-linkthe CD3 complexes on T cells. The Fc portion of mabs of murine origin isalso the main reason of anti-globulin response. The antibody of thepresent invention is devoid of the immunoglobulin constant domains and,therefore, is not able to interact with FcRs and is also much lessimmunogenic.

The antibodies of the present invention can be prepared by methods knownto the person skilled in the art, e.g. by the following methods:

(a) Construction of single chain Fv-antibodies by combining the genesencoding at least two immunoglobulin variable V_(H) and V_(L) domains,either separated by peptide linkers or by no linkers, into a singlegenetic construct and expressing it in bacteria or other appropriateexpression system.

(b) Non-covalent dimerization or multimerization of single chainFv-antibodies comprising at least two V_(H) and V_(L) specific to humanCD3 either separated by peptide linkers or by no linkers, in anorientation preventing their intramolecular pairing.

The term “capable of suppressing an immune reaction” means that theantibody is able, on the one hand, to prevent activation of Tlymphocytes by foreign alloantigen and, on the other hand, toselectively deplete already activated T cells.

The antibody of the present invention may be a monovalent, bivalent ormultivalent antibody.

In a preferred embodiment, the antibody of the present invention is anon-covalent dimer of a single-chain Fv-antibody (scFv) (“diabody” ; seeFIG. 1) comprising CD3-specific V_(H) and V_(L) domains, eitherseparated by peptide linkers or by no linkers.

In a further preferred embodiment, the antibody of the present inventioncomprises two single-chain Fv-antibodies (scFv) (see FIG. 1) comprisingCD3-specific V_(H) and V_(L) domains.

In a further preferred embodiment, the antibody of the present inventionis a single chain diabody (see FIG. 1) comprising CD3-specific V_(H) andV_(L) domains.

The term “Fv-antibody” as used herein relates to an antibody containingvariable domains but not constant domains. The term “peptide linker” asused herein relates to any peptide capable of connecting two variabledomains with its length depending on the kinds of variable domains to beconnected. The peptide linker might contain any amino acid residue,although the amino acid combinations SAKTTP or SAKTTPKLGG are preferred.The peptide linker connecting single scFv of (scFv)₂ and single chaindiabodies (scDb) might contain any amino acid residue, althoughone-to-three repeats of amino acid combination GGGGS are preferred for(scFv)₂ and three-to-four repeats of GGGGS are preferred for scDb.

In a more preferred embodiment, the antibody of the present inventioncontains variable domains substantially corresponding to the variabledomains of the antibody produced by the hybridoma of ATCC deposit numberCRL 8001.

In an even more preferred embodiment, the antibody of the presentinvention is characterized in that a cysteine at position H100A (Kabatnumbering system) has been replaced by another amino acid, preferably bya serine.

The present invention also relates to a polynucleotide encoding anantibody of the present invention and vectors, preferably expressionvectors containing said polynucleotides. The recombinant vectors can beconstructed according to methods well known to the person skilled in theart; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, ColdSpring Harbor Laboratory (1989) N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding the antibody of the present invention.These include, but are not limited to, microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid, or cosmid DNAexpression vectors; yeast transformed with yeast expression vectors;insect cell systems infected with virus expression vectors (e.g.,baculovirus); plant cell systems transformed with virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids);or animal cell systems.

The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector-enhancers, promoters, 5′- and b3′-untranslated regions which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the Bluescript.RTM. phagemid (Stratagene,Lajolla, Calif.) or pSportl.TM. plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding themultivalent multimeric antibody, vectors based on SV40 or EBV may beused with an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the antibody of the presentinvention. Vectors suitable for use in the present invention include,but are not limited to the pSKK expression vector for expression inbacteria.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used; for reviews, see Grant et al. (1987)Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding the antibody of the present invnetion may be drivenby any of a number of promoters. For example, viral promoters such asthe 35S and 19S promoters of CaMV may be used alone or in combinationwith the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.6:307-311). Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. and Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

An insect system may also be used to express the antibodies of thepresent invention. For example, in one such system, Autographacalifornica nuclear polyhedrosis virus (AcNPV) is used as a vector toexpress foreign genes in Spodoptera frugiperda cells or in Trichoplusialarvae. The sequences encoding said antibodies may be cloned into anon-essential region of the virus, such as the polyhedrin gene, andplaced under control of the polyhedrin promoter. Successful insertion ofthe gene encoding said antibody will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which APOP may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding an antibody of the present invention may beligated into an adenovirus transcription/translation complex consistingof the late promoter and tripartite leader sequence. Insertion in anon-essential E1 or E3 region of the viral genome may be used to obtaina viable virus which is capable of expressing the antibody in infectedhost cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci.81:3655-3659). In addition, transcription enhancers, such as the Roussarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding the antibody of the present invention.Such signals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the antibody, its initiation codon, andupstream sequences are inserted into the appropriate expression vector,no additional transcriptional or translational control signals may beneeded. However, in case where only coding sequence is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed antibody chains in the desired fashion. Post-translationalprocessing which cleaves a “prepro” form of the protein may also be usedto facilitate correct insertion, folding and/or function. Different hostcells which have specific cellular machinery and characteristicmechanisms for post-translational activities (e.g., CHO, HeLa, MDCK,HEK293, and W138), are available from the American Type CultureCollection (ATCC; Bethesda, Md.) and may be chosen to ensure the correctmodification and processing of the foreign antibody chains.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines which stably expressthe antibody may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.- cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, beta-glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

A particular preferred expression vector is pSKK3-scFv6_(—anti-CD)3deposited with the DSMZ (Deutsche Sammlung für Mikroorganismen undZellen) according to the Budapest Treaty under DSM 15137 on Aug. 16,2002.

The present invention also relates to a composition containing anantibody, polynucleotide or an expression vector of the presentinvention. Preferably, said composition is a pharmaceutical compositionpreferably combined with a suitable pharmaceutical carrier. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc.. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Administration of thesuitable compositions may be effected by different ways, e.g. byintravenous, intraperetoneal, subcutaneous, intramuscular, topical orintradermal administration. The route of administration, of course,depends on the kind of therapy and the kind of compound contained in thepharmaceutical composition. The dosage regimen will be determined by theattending physician and other clinical factors. As is well known in themedical arts, dosages for any one patient depends on many factors,including the patient's size, body surface area, age, sex, theparticular compound to be administered, time and route ofadministration, the kind of therapy, general health and other drugsbeing administered concurrently.

A preferred medical use of the compounds of the present inventiondescribed above is immunotherapy, preferably a therapy against acutetransplant rejections and possibly against autoimmune diseases, such astype I diabetes, multiple sclerosis and rheumatoid arthritis.

The examples below explain the invention in more detail.

EXAMPLE 1 Construction of the Plasmids pHOG-scFv10/anti-CD3,pHOG-scFv6/anti-CD3, pSKK3-scFv10/anti-CD3 and pSKK3-scFv6/anti-CD3 forthe expression of anti-CD3 scFv₁₀ and scFv₆ antibodies in bacteria

For constructing the genes encoding the anti-CD3 scFv₁₀ and scFv₆ (FIG.2), the plasmid pHOG21-dmOKT3 containing the gene for anti-human CD3scFv₁₈ (Kipriyanov et al., 1997, Protein Engineering 10, 445-453) wasused. To facilitate the cloning procedures, NotI restriction site wasintroduced into the plasmid pHOG21-dmOKT3 by PCR amplification of scFv₁₈gene using primers Bi3sk, 5′-CAGCCGGCCATGGCGCAGGTGCAACTGCAGCAG andBi9sk, 5′-GAAGATGGATCCAGCGGCCGCAGTATCAGCCCGGTT. The resulting 776 bp PCRfragment was digested with NcoI and NotI and cloned into theNcoI/NotI-linearized vector pHOG21-CD19 (Kipriyanov et al., 1996, J.Immunol. Methods 196, 51-62), thus generating the plasmidpHOG21-dmOKT3+Not. The gene coding for OKT3 V_(H) domain with a Cys-Sersubstitution at position 100A according to Kabat numbering scheme(Kipriyanov et al., 1997, Protein Engineering 10, 445-453) was amplifiedby PCR with primers DP1, 5′-TCACACAGAATTCTTAGATCTATTAAAGAGGAGAAATTAACCand either DP2, 5′-AGCACACGATATCACCGCCAAGCTTGGGTGTTGTTTTGGC or OKT_(—)5,5′- TATTAAGATATCGGGTGTTGTTTTGGCTGAGGAG, to generate the genes for V_(H)followed by linkers of 10 and 6 amino acids, respectively (FIG. 2). Theresulting 507 bp and 494 bp PCR fragments were digested with NcoI andEcoRV and cloned into NcoI/EcoRV-linearized plasmid pHOG21-dmOKT3+Not,thus generating the plasmids pHOG21-scFv10/anti-CD3 andpHOG21-scFv6/anti-CD3, respectively.

To increase the yield of functional scFv-antibodies in the bacterialperiplasm, an optimized expression vector pSKK3 was generated (FIG. 3).This vector was constructed on the basis of plasmid PHKK (Horn et al.,1996, Appl. Microbiol. Biotechnol. 46, 524-532) containing hok/sokplasmid-free cell suicide system (Thisted et al., 1994, EMBO J. 13,1960-1968). First, the gene coding for hybrid scFv VH³-V_(L) 19 wasamplified by PCR from the plasmid pHOG3-19 (Kipriyanov et al., 1998,Int. J. Cancer 77, 763-772) using the primers 5-NDE, 5′-GATATACATATGAAATACCTATTGCCTACGGC, and 3-AFL,5′-CGAATTCTTAAGTTAGCACAGGCCTCTAGAGACACACAGATCTTTAG. The resulting 921 bpPCR fragment was digested with NdeI and AflII and cloned into theNdeI/AflII linearized plasmid pHKK generating the vector pHKK3-19. Todelete an extra XbaI site, a fragment of pHKK plasmid containing3′-terminal part of the lacI gene (encoding the lac repressor), thestrong transcriptional terminator tHP and wild-type lacpromoter/operator was amplified by PCR using primers 5-NAR,5′-CACCCTGGCGCCCAATACGCAAACCGCC, and 3-NDE,5′-GGTATTTCATATGTATATCTCCTTCTTCAGAAATTCGTAATCATGG. The resulting 329 bpDNA fragment was digested with NarI and NdeI and cloned intoNarI/NdeI-linearized plasmid pHKK3-19 generating the vector pHKK□OXba.To introduce a gene encoding the Skp/OmpH periplasmic factor for higherrecombinant antibody production (Bothmann and Pluckthun, 1998, Nat.Biotechnol. 16, 376-380), the skp gene was amplified by PCR with primersskp-3, 5′-CGAATTCTTAAGAAGGAGATATACATATGAAAAAGTGGTTATTAGCTGCAGG andskp-4, 5′-CGAATTCTCGAGCATTATTTAACCTGTTTCAGTACGTCGG using as a templatethe plasmid pGAH317 (Holck and Kleppe, 1988, Gene 67, 117-124). Theresulting 528 bp PCR fragment was digested with AflII and XhoI andcloned into the AflII/XhoI digested plasmid pHKK□Xba resulting in theexpression plasmid pSKK2. For removing the sequence encoding potentiallyimmunogenic c-myc epitope, the NcoI/XbaI-linearized plasmid pSKK2 wasused for cloning the NcoI/XbaI-digested 902 bp PCR fragment encoding thescFv phOx31E (Marks et al. 1997, BioTechnology 10, 779-783), which wasamplified with primers DP1 and His-Xba,5′-CAGGCCTCTAGATTAGTGATGGTGATGGTGATGGG. The resulting plasmid pSKK3 wasdigested with NcoI and NotI and used as a vector for cloning the genescoding for anti-CD3 scFv₆ and scFv₁₀ , that were isolated as 715 bp and727 bp DNA fragments after digestion of plasmids pHOG21-scFv6/anti-CD3and pHOG21-scFv10/anti-CD3, respectively, with NcoI and NotI.

The generated plasmids pSKK3-scFv6/anti-CD3 (FIG. 3) andpSKK3-scFv10/anti-CD3 contain several features that improve plasmidperformance and lead to increased accumulation of functional bivalentproduct in the E. coli periplasm under conditions of both shake-flaskcultivation and high cell density fermentation. These are the hok/sokpost-segregation killing system, which prevents plasmid loss, strongtandem ribosome-binding sites and a gene encoding the periplasmic factorSkp/OmpH that increases the functional yield of antibody fragments inbacteria. The expression cassette is under the transcriptional controlof the wt lac promoter/operator system and includes a short sequencecoding for the N-terminal peptide of β-galactosidase (lacZ') with afirst rbs derived from the E. coli lacZ gene, followed by genes encodingthe scFv-antibody and Skp/OmpH periplasmic factor under thetranslational control of strong rbs from gene 10 of phage T7 (T7g10).Besides, the gene of scFv-antibody is followed by a nucleotide sequenceencoding six histidine residues for both immunodetection andpurification of recombinant product by immobilized metal-affinitychromatography (IMAC).

EXAMPLE 2 Production in Bacteria and Purification of scFv-Antibodies

The E. coli K12 strain RV308 (Δlacχ74 galISII::OP308strA) (Maurer etal., 1980, J. Mol. Biol. 139, 147-161) (ATCC 31608) was used forfunctional expression of scFv-antibodies. The bacteria transformed withthe expression plasmids pSKK3-scFv6/anti-CD3 and pSKK3-scFv10/anti-CD3,respectively, were grown overnight in 2xYT medium with 100 μg/mlampicillin and 100 mM glucose (²xYTGA) at 26° C. The overnight cultureswere diluted in fresh 2xYTGA medium till optical density at 600 nm(OD₆₀₀) of 0.1 and continued to grow as flask cultures at 26° C. withvigorous shaking (180-220 rpm) until OD₆₀₀ reached 0.6-0.8. Bacteriawere harvested by centrifugation at 5,000 g for 10 min at 20° C. andresuspended in the same volume of fresh YTBS medium (2xYT containing 1 Msorbitol, 2.5 mM glycine betaine and 50 μg/ml ampicillin).Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a finalconcentration of 0.2 mM and growth was continued at 21° C. for 14-16 h.Cells were harvested by centrifugation at 9,000 g for 20 min at 4° C. Toisolate soluble periplasmic proteins, the pelleted bacteria wereresuspended in 5% of the initial volume of ice-cold 200 mM Tris-HCl, 20%sucrose, 1 mM EDTA, pH 8.0. After 1 h incubation on ice with occasionalstirring, the spheroplasts were centrifuged at 30,000 g for 30 min and4° C. leaving the soluble periplasmic extract as the supernatant andspheroplasts plus the insoluble periplasmic material as the pellet. Theperiplasmic extract was thoroughly dialyzed against 50 mM Tris-HCl, 1 MNaCl, pH 7.0, and used as a starting material for isolatingscFv-antibodies. The recombinant product was concentrated by ammoniumsulfate precipitation (final concentration 70% of saturation). Theprotein precipitate was collected by centrifugation (10,000 g, 4° C., 40min) and dissolved in 10% of the initial volume of 50 mM Tris-HC1, 1 MNaCl, pH 7.0, followed by thorough dialysis against the same buffer.Immobilized metal affinity chromatography (IMAC) was performed at 4° C.using a 5 ml column of Chelating Sepharose (Amersham Pharmacia,Freiburg, Germany) charged with Cu²⁺and equilibrated with 50 mMTris-HCl, 1 M NaCl, pH 7.0 (start buffer). The sample was loaded bypassing the sample over the column by gravity flow. The column was thenwashed with twenty column volumes of start buffer followed by startbuffer containing 50 mM imidazole until the absorbance (280 nm) of theeffluent was minimal (about thirty column volumes). Absorbed materialwas eluted with 50 mM Tris-HCl, 1 M NaCl, 300 mM imidazole, pH 7.0, as 1ml fractions. The eluted fractions containing recombinant protein wereidentified by reducing 12% SDS-PAGE followed by Coomassie staining. Thepositive fractions were pooled and subjected to buffer exchange for 50mM imidazole-HCl_(1,) 50 mM NaCl (pH 7.0) using pre-packed PD-10 columns(Pharmacia Biotech, Freiburg, Germany). The turbidity of proteinsolution was removed by centrifugation (30,000 g, 1 h, 4° C).

The final purification was achieved by ion-exchange chromatography on aMono S HR 5/5 column (Amersham Pharmacia, Freiburg, Germany) in 50 mMimidazole-HCl, 50 mM NaCl, pH 7.0, with a linear 0.05-1 M NaCl gradient.The fractions containing scFv-antibody were concentrated withsimultaneous buffer exchange for PBS containing 50 mM imidazole, pH 7.0(PBSI buffer), using Ultrafree-15 centrifugal filter device (Millipore,Eschborn, Germany). Protein concentrations were determined by theBradford dye-binding assay (Bradford, 1976, Anal. Biochem., 72, 248-254)using the Bio-Rad (Munich, Germany) protein assay kit. SDS-PAGE analysisdemonstrated that anti-CD3 scFv₁₀ and scFv₆ migrated as single bandswith a molecular mass (M_(r)) around 30 kDa (FIG. 4). Size-exclusionchromatography on a calibrated Superdex 200 HR 10/30 column (AmershamPharmacia) demonstrated that scFv₆ was mainly in a dimeric form with Mraround 60 kDa, while scFv₁₀ was pure monomer (FIG. 5).

EXAMPLE 3 Cell Binding Measurements

The human CD3⁺T-cell leukemia cell line Jurkat was used for flowcytometry experiments. The cells were cultured in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mML-glutamine, 100 U/mL penicillin G sodium and 100 μg/ml streptomycinsulfate (all from Invitrogen, Groningen, The Netherlands) at 37° C. in ahumidified atmosphere with 5% CO_(2.) 1×10⁶ cells were incubated with0.1 ml phosphate buffered saline (PBS, Invitrogen, Groningen, TheNetherlands) supplemented with 2% heat-inactivated fetal calf serum(FCS, Invitrogen, Groningen, The Netherlands) and 0.1% sodium azide(Roth, Karlsruhe, Germany) (referred to as FACS buffer) containingdiluted scFv-antibodies or mAb OKT3 (Orthoclone OKT3, Cilag, Sulzbach,Germany) for 45 min on ice. After washing with FACS buffer, the cellswere incubated with 0.1 ml of 0.01 mg/ml anti-(His)₆ mouse mAb13/45/31-2 (Dianova, Hamburg, Germany) in the same buffer for 45 min onice. After a second washing cycle, the cells were incubated with 0.1 mlof 0.015 mg/ml FITC-conjugated goat anti-mouse IgG (Dianova, Hamburg,Germany) under the same conditions as before. The cells were then washedagain and resuspended in 0.5 ml of FACS buffer containing 2 μg/mlpropidium iodide (Sigma-Aldrich, Taufkirchen, Germany) to exclude deadcells. The fluorescence of 1×10⁴ stained cells was measured using aBeckman-Coulter Epics XL flow cytometer (Beckman-Coulter, Krefeld,Germany). Mean fluorescence (F) was calculated using System-II andExpo32 software (Beckman-Coulter, Krefeld, Germany) and the backgroundfluorescence was subtracted. Equilibrium dissociation constants (K_(d))were determined by fitting the experimental values to theLineweaver-Burk equation: 1/F=1/F_(max),+(K_(d)/F_(max))(1/[Ab]) usingthe software program PRISM (GraphPad Software, San Diego, Calif.).

The flow cytometry experiments demonstrated a specific interaction ofscFv-antibodies to Jurkat cells expressing CD3 on their surface. Thefluorescence intensities obtained for scFv₆ were significantly higherthan for scFv₁₀ reflecting the 10-fold difference in affinity values forthese two scFv-antibodies (FIG. 6, Table 1). The deduced affinity valuefor scFv₆ was fairly close to that of mAb OKT3 thus confirming thebivalent binding of scFv6 to the cell surface.

EXAMPLE 4 In Vitro Cell Surface Retention

To investigate the biological relevance of the differences betweenscFv₆, scFv₁₀ and OKT3 in direct binding experiments, the in vitroretention of the scFv-antibodies on the surface of CD3⁺Jurkat cells wasdetermined by flow cytometry (FIG. 7). Cell surface retention assayswere performed at 37° C. under conditions preventing internalization ofcell surface antigens, as described (Adams et al., 1998, Cancer Res. 58,485-490), except that the detection of retained scFv-antibodies wasperformed using mouse anti-(His)₆ mAb 13/45/31-2 (0.01 mg/ml; Dianova,Hamburg, Germany) followed by FITC-conjugated goat anti-mouse IgG (0.015mg/ml; Dianova, Hamburg, Germany). Kinetic dissociation constant(k_(off)) and half-life (t_(½)) values for dissociation of antibodieswere deduced from a one-phase exponential decay fit of experimental datausing the software program PRISM (GraphPad Software, San Diego, Calif).The monovalent scFv₁₀ had a relatively short retention half-life (1.02min), while scFv₆ and OKT3 had 1.5-fold and 2.5-fold longert_(½)respectively, thus correlating well with their higher bindingaffinities deduced from the direct binding experiments (FIG. 7, Table1). TABLE 1 Affinity and kinetics of anti-CD3 antibodies binding to CD3⁺Jurkat cells Antibody K_(d) (nM) k_(off) (s⁻¹/10⁻³) t_(1/2) (min) mAbOKT3 2.06 4.47 2.59 scFv₆ 4.58 7.82 1.48 scFv₁₀ 51.92 11.33 1.02The dissociation constants (K_(d)) were deduced from Lineweaver-Burkplots shown in FIG. 6. The k_(off) values were deduced from Jurkat cellsurface retention experiments shown in FIG. 7. The half-life values(t_(½)) for dissociation of antibody-antigen complexes were deduced fromthe ratio ln2/k_(off).

EXAMPLE 5 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)

Human PBMCs were isolated from the heparinized peripheral blood ofhealthy volunteers by density gradient centrifugation. The blood sampleswere twice diluted with PBS (Invitrogen, Groningen, The Netherlands),layered on a cushion of Histopaque-1077 (Sigma-Aldrich, Taufkirchen,Germany) and centrifuged at 800 g for 25 min. The PBMCs located in theinterface were collected and washed three times with PBS before use.

EXAMPLE 6 Cell Proliferation Assay

Isolated PBMCs were resuspended in RPMI 1640 medium supplemented with10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin G sodiumsalt and 0.1 mg/ml streptomycin sulfate (all from Invitrogen, Groningen,The Netherlands) and placed to 96-well flat-bottom tissue culture plates(Greiner, Frickenhausen, Germany) at a density of 2×10⁵ cells per well.Triplicates of cultures were incubated with serial dilutions of solubleantibodies at 37° C. in a humidified atmosphere containing 5% CO₂ forthe indicated time followed by 18 h pulsing with 0.01 mM5-bromo-2′-deoxyuridine (BrdU). Incorporation of BrdU was determined byCell Proliferation ELISA (Roche, Mannheim, Germany) according to themanufacturers instructions.

During incubation for 24-36 h, neither scFv₆ nor scFv₁o inducedproliferation of both autologous (donor A alone and donor B alone,respectively) and mixed lymphocyte cultures (donor A+B). In contrast,mAb OKT3 demonstrated high mitogenic activity for all tested 24 hcultures, obviously due to CD3-crosslinking via FcyR-bearing cells (FIG.8).

The OKT3-induced T-cell proliferation was significantly higher inautologous PBMC cultures incubated for 72-90 h, while scFv₆ and scFv₁₀demonstrated only minor effects in comparison with 24-h incubation (FIG.9). In mixed PBMC cultures (donor A+B) incubated for 72 h withoutantibody treatment, a mixed lymphocyte reaction (MLR) developed.Treatment of mixed PBMC cultures with OKT3 had no effect on MLR, whileboth scFv-antibodies were able to suppress MLR in aconcentration-dependent manner, thus reaching the background level at aconcentration of 10 μg/ml (FIG. 9).

EXAMPLE 7 Analyses of Cytokine Release

For measurement of cytokine secretion by activated lymphocytes, 2×10⁶PBMCs were plated in individual wells of 24-well plates (Greiner,Frickenhausen, Germany) in RPMI 1640 medium supplemented with 10%heat-inactivated FCS, 2 mM L-glutamine, b 100 U/ml penicillin G sodiumsalt and 0.1 mg/ml streptomycin sulfate (all from Invitrogen, Groningen,The Netherlands) together with the indicated antibodies. Fordetermination of secretion of IL-2, TNF-α and IFN-γ, aliquots of theculture supernatants were collected after 24 h, 36 h and 72 h,respectively. Cytokine levels were measured in duplicates using thecommercially available ELISA kits for IL-2 (Pharmingen, San Diego,Calif.), TNF-α and IFN-γ (Endogen, Cambridge, Mass.).

In both autologous and mixed PBMC cultures, OKT3 induced a strongrelease of IL-2 (FIG. 10), IFN-γ(FIG. 11) and TNF-α (FIG. 12). Incontrast, the autologous PBMC cultures treated with scFv₆ and scFv₁₀,respectively, did not produce IL-2 (FIG. 10), IFN-γ (FIG. 11) and TNF-α(FIG. 12). Mixed lymphocyte cultures incubated without antibodiesdemonstrated release of significant amounts of cytokines as a result ofallogeneic stimulation. This secretion of IL-2 and IFN-γ could besuppressed by scFv-antibodies in a dose-dependent manner (FIGS. 10 and11). Bivalent scFv₆ demonstrated approximately tenfold higher efficacythan scFv₁₀. In contrast, mAb OKT3 had rather induction than suppressionof cytokine release in mixed PBMC cultures.

EXAMPLE 8 Alteration of Surface Antigens on PBMCs Treated with Anti-CD3Antibodies

For determination the cell surface expression of the alpha-subunit ofIL-2 receptor (CD25) as an early activation marker, 2×10⁶ PBMCs wereplated in individual wells of 24-well plates (Greiner, Frickenhausen,Germany) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS,2 mM L-glutamine, 100 U/ml penicillin-G sodium salt and 0.1 mg/mlstreptomycin sulfate (all from Invitrogen, Groningen, The Netherlands)together with the indicated antibodies. The cells were harvested after90 h incubation and stained for flow cytometric analysis withPE-conjugated anti-CD25 mAb B1.49.9 and with the corresponding isotypecontrols (all from Beckman-Coulter, Krefeld, Germany), as described inExample 3. 10⁴ lymphocytes were analyzed with a Beckman-Coulter Epics XLflow cytometer (Beckman-Coulter, Krefeld, Germany). Mean fluorescence(F) was calculated using System-II software (Beckman-Coulter, Krefeld,Germany), and background fluorescence was subtracted.

PBMCs that were cultured in the presence of OKT3 showed a strongupregulation of the early activation marker IL-2R□ (CD25) on theirsurface, as determined by flow cytometry (FIG. 12). In contrast, none ofthe PBMC cultures treated either with scFv₆ or scFv₁₀ showed elevatedlevels of CD25 expression (FIG. 13). Thus, these results clearlydemonstrate that, unlike mAb OKT3, scFv₆ and scFv₁₀ do not posses theT-cell activating properties.

EXAMPLE 9 Modulation and Coating of TCR/CD3 on Lymphocytes treated withAnti-CD3 Antibodies

To measure the modulation and coating of cell surface TCR/CD3 onlymphocytes, 2×10⁶ PBMCs were plated in individual wells of 24-wellplates (Greiner, Frickenhausen, Germany) in RPMI 1640 mediumsupplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/mlpenicillin-G sodium salt and 0.1 mg/ml streptomycin sulfate (all fromInvitrogen, Groningen, The Netherlands) together with the indicatedantibodies. The cells were harvested after 24 h incubation and stainedfor flow cytometric analysis with FITC-conjugated OKT3 (Dr. Moldenhauer,German Cancer Research Center, Heidelberg) or PC5-conjugated anti-TCRα/β(Beckman-Coulter, Krefeld, Germany) and the corresponding isotypecontrols (Beckman-Coulter, Krefeld, Germany). The cells werecounterstained with anti-CD5 antibodies (Beckman-Coulter, Krefeld,Germany) for T lymphocytes and analyzed with a Beckman-Coulter Epics XLflow cytometer (Beckman-Coulter, Krefeld, Germany). Mean fluorescence(F) of OKT3-FITC and TCR-PC5 from CD5-positive cells was calculatedusing System-II software (Beckman-Coulter, Krefeld, Germany).Calculation of CD3 modulation and coating was performed as describedpreviously (Cole, M.S. et al., 1997, J. Immunol. 159, 3613-3621):${\%\quad{CD}\quad 3\quad{modulation}} = {\frac{{{untreated}\quad{cells}\quad{F\left( {{anti}\text{-}{TCR}} \right)}} - {{treated}\quad{cells}\quad{F\left( {{anti}\text{-}{TCR}} \right)}}}{{untreated}\quad{cells}\quad{F\left( {{anti}\text{-}{TCR}} \right)}} \times 100}$${\%\quad{CD}\quad 3\quad{coating}} = {\frac{{treated}\quad{cells}\quad{F\left( {{anti}\text{-}{TCR}} \right)}}{{control}\quad{cells}\quad{F\left( {{anti}\text{-}{TCR}} \right)}} - {\frac{{treated}\quad{cells}\quad{F\left( {{OKT}\quad 3} \right)}}{{control}\quad{{cells}\left( {{OKT}\quad 3} \right)}} \times 100}}$Coating, which is defined as the number of CD3 molecules on the surfaceof T lymphocytes that are antibody bound and therefore not detectable byFITC-conjugated mAb OKT3, was only observed in one experiment with thelowest concentration of OKT3 and anti-CD3 scFv₆ (FIG. 14). CD3modulation, which represents the fraction of TCR/CD3 complexes on thesurface of T cells that is lost after antibody treatment, is efficiently(>90%) induced by mAb OKT3 and anti-CD3 scFv₆ at concentrations in therange between 0.1 μg/ml and 10 μg/ml (FIG. 14). In contrast, themodulation activity of anti-CD3 scFv₁₀ was much lower and could beobserved only at concentrations above 1 μg/ml (FIG. 14).

1. A bivalent or multivalent antibody characterized by the followingfeatures: (a) it is capable of supressing an immune reaction; (b) it isdevoid of constant antibody regions; and (c) it binds an epitope on theCD3 complex of the T-cell receptor.
 2. The antibody of claim 1 that is adiabody.
 3. The antibody of claim 1 that comprises two scFv antibodieslinked by a peptide linker.
 4. The antibody of claim 1 that is a singlechain diabody.
 5. The antibody according to claim 1, wherein variableV_(H) and V_(L) domains are connected via the peptide linker SAKTTP orSAKTTPKLGG.
 6. The antibody according to claim 1, wherein its variabledomains correspond to the variable domains of an antibody produced bythe hybridoma of ATCC deposit number CRL
 8001. 7. The antibody accordingto claim 6, wherein a cysteine at position H100A has been exchanged foranother amino acid.
 8. The antibody according to claim 7, wherein thecysteine has been exchanged for a serine.
 9. A polynucleotide, whichencodes an antibody according to claim
 1. 10. An expression vectorcomprising the polynucleotide of claim
 9. 11. The expression vector ofclaim 10, which is pSKK3-scFv_(—)6-anti-CD3 (DSM 15137).
 12. A host cellcontaining the expression vector of claim 10 or
 11. 13. A pharmaceuticalcomposition comprising the antibody of claim 1, the polynucleotide ofclaim 9, or the expression vector of claim
 10. 14. A method forimmunotherapy comprising the step of administering to a subject thepharmaceutical composition according to claim
 13. 15. A method forimmunotherapy comprising the step of administering to a subject apharmaceutical composition comprising the antibody of claim
 1. 16. Themethod according to claim 14 , wherein said immunotherapy is a therapyagainst acute transplant rejections.
 17. A method for gene therapycomprising the step of administering to a subject a pharmaceuticalcomposition comprising the polynucleotide of claim 9 or the expressionvector of claim 10.